Multidrug Resistance Tuberculosis in the Context of Co-Infection in Ethiopia: A Systematic Review and Meta-Analysis

Abstract

The rise of multidrug-resistant tuberculosis (MDR-TB) remains a critical public health challenge, particularly in developing countries like Ethiopia. This systematic review and meta-analysis aimed to estimate the pooled prevalence of MDR-TB with co-infections and assess its effects among different co-infections in Ethiopia. The systematic review and meta-analysis were conducted from August to October 2024. The study adhered to PRISMA guidelines and utilized various academic databases including PubMed, Web of Science and Science Direct to identify relevant articles. To check for publication bias and small study effects, a funnel plot and Egger’s test were employed. The statistical analysis was performed with R software version 4.4.1. From an original pool of 6,461 papers, 15 studies published between 2014 and 2024 were considered after applying certain inclusion and exclusion criteria. The analysis revealed an overall pooled prevalence of MDR-TB in the context of co-infections at 20% (95% CI: 14.0–26.0). Notably, the prevalence was higher among individuals with HIV co-infection at 23.2% (95% CI: 18.3–28.0), while it was lower in those with diabetes co-infection at 10% (95% CI: 3.0-17.3). The study found significant heterogeneity among the reported prevalence rates (I² = 94.93%, p < 0.001). These findings highlight the complex interplay between MDR-TB and other co-infections, posing significant challenges for clinical management and public health in Ethiopia. To enhance health outcomes and curb the spread of MDR-TB, government and public health authorities must implement targeted interventions, including monitoring and treatment programs in high-prevalence areas.

Supplementary Information

The online version contains supplementary material available at 10.1007/s44197-025-00360-7.

Introduction

Mycobacterium tuberculosis, the bacteria that causes tuberculosis (TB), is still a serious global health concern that affects millions of people annually and results in morbidity and mortality. Every year, TB affects the health of over 10.4 million people worldwide, leading to about 1.8 million TB-related fatalities; the bulk (95%) of these deaths were reported from nations with poor resources [1, 2]. Tuberculosis is one of zoonotic chronic respiratory infectious illness. Goats, sheep, and cattle are examples of domestic animals that are significant but often overlooked TB hosts. The spread of tuberculosis poses a major risk to the health of humans and animals [3–6]. Treatment can be divided into first line and second line drugs according to the WHO TB treatment regime [7]. First line drugs include Isoniazid (INH), Rifampin (RIF), pyrazinamide (PZA) and ethambutol (EMB). Similarly, the second-line drugs, including bedaquiline (Bdq), linezolid (Lzd), moxifloxacin (Mfx), levofloxacin (Lfx), clofazimine (Cfz), cycloserine (Cs), para-aminosalicylic acid (PAS), propylthiouracil, and amikacin (Am) [8, 9]. Resistance to first line anti-TB drugs has been linked to mutations in at least ten genes [10].

Drug-resistant TB (DR-TB) occurs when tuberculosis develops resistance to one or more of the four first-line antibiotics, with isoniazid being the most commonly occurred resistant [11]. The most concerning component of the antibiotic resistance pandemic is multidrug-resistant tuberculosis (MDR-TB), which is caused by Mycobacterium tuberculosis resistant to both isoniazid and rifampicin with or without resistance to other medications [12]. MDR-TB’s rise continues to pose a severe threat to public health, especially in developing nations [13]. The global prevalence of MDR-TB is estimated to be around 73% [14].

Ethiopia was included among 30 high TB burdened countries in the globe. According to the World Health Organization (WHO) global TB report, 117,705 cases and 28,600 deaths due to TB have been reported in the country [14]. The overall TB detection rate was 11.7% [15]. The epidemiology of MDR-TB in Ethiopia showed that 2.18% of newly diagnosed and 21.07% of previously treated cases had MDR-TB [16].

Tuberculosis is the main cause of widespread mortality, especially among people living with Human immunodeficiency virus (HIV) and other co-infections [17]. Co-infections that occur along with MDR-TB also increase risk of transmission as they have a higher bacterial load and longer periods of infectiousness, which can increase the transmission of MDR-TB within communities [18]. Furthermore, co-infections can complicate the treatment of MDR-TB by requiring adjustments to the standard treatment regimen. These complications often lead to longer treatment durations, increased side effects, and lower treatment success rates [19]. Co-infections are associated with worse treatment outcomes, including higher rates of morbidity and mortality [20]. Understanding co-infections is essential for creating more effective treatment protocols considering the complexities of managing multiple conditions [21].

In Ethiopia, a sectional cross-sectional study was done to explore the prevalence and associated variables of MDR-TB/HIV co-infection in central Ethiopia [22, 23]. Among the MDR-TB 196 cases, 128 cases were MDR-TB/HIV co-infected [24]. Previous studies have shown that MDR-TB in the context of co-infections is a serious condition in Ethiopia [23, 25–28]. Although several studies have examined the prevalence and risk factors associated with MDR-TB over the past years, comprehensive country level estimates of MDR-TB in the context of co-infections and comparison estimates among co-infections are lacking. Therefore, this systematic review and meta-analysis are used to estimate the pooled prevalence of MDR-TB in the context of co-infections and to compare the pooled prevalence of MDR-TB among different coinfections in Ethiopia.

Methodology

Study Protocols

This systematic review and meta-analysis were conducted from August 2024 to October 2024 according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [29] (Supplementary File 1). The PRISMA checklist was used to ensure all relevant information was included in the analysis. The pooled prevalence of MDR-TB in the context of co-infections and estimates of the pooled prevalence of MDR-TB among different subgroups were the outcomes of interest.

Description of Study Setting

The current systematic review and meta-analysis were performed in Ethiopia. Ethiopia is situated between latitudes 3° and 15° North and longitudes 33° and 48° East in the North Eastern region of Africa. The nation, which is the ninth largest and second most populous in Africa, is roughly 1.13 million km³ in size, of which 1.12 million km³ are on land, and the rest, 7444 km³ are covered in water (rivers, lakes, ponds, etc.). Ethiopia is the second most populated nation in Africa, behind Nigeria [30]. Ethiopia, a nation with a high rate of tuberculosis, has difficulties in controlling the disease because of its diversified population, which is dispersed over both urban and rural areas, its poor healthcare infrastructure, and its varied socio-economic circumstances [31]. This systematic review uses data from studies done all over Ethiopia to evaluate the prevalence and risk factors associated with multidrug-resistant tuberculosis (MDR-TB) in these co-infected populations, considering the impact of MDR-TB and the prevalence of HIV and diabetes in the area.

Literature Search Strategy

Academic databases such as PubMed, PubMed Central, Science Direct, Web of Science, and Google Scholar were used to search for articles focusing on MDR-TB in the context of co-infections in Ethiopia. The language of publication is restricted to English. The year was restricted from 2014 to 2024. The article search was conducted using various MeSH (medical subject heading) terms, including: ‘Prevalence’ OR ‘Epidemiology’ AND ‘MDR-TB’ OR ‘Multidrug resistant tuberculosis’ AND ‘Co-infections’ OR ‘HIV’ OR ‘Viral hepatitis’ OR ‘Diabetes’ OR ‘COVID 19’ AND ‘Ethiopia’. Mendeley version 1.19.8 (Mendeley Ltd) software was used to remove duplication of articles.

Inclusion and Exclusion Criteria

Regardless of the research population type, articles reporting MDR-TB in the context of co-infections were collected. The study’s objectives served as the basis for defining the inclusion and exclusion criteria. The inclusion criteria include articles that focused on MDR-TB and co-infections, written in English, published between 2014 and 2024, using a cross-sectional study design, and full-text articles included in the systematic review and meta-analysis. Papers are rejected based on the exclusion criteria. These criteria include articles that were unpublished, systematic reviews and meta-analyses, studies that focus solely on an MDR-TB, excluding co-infections, did not align with the specified emphasis outlined in the systematic review, exclude important things about prevalence, not studied in Ethiopia and not written in English.

Data Extraction

Data entered in suitable formats into Mendeley reference manager software version 1.19.8 (Mendeley Ltd) came from various academic databases such as PubMed, PubMed Central, Science Direct, Web of Science, and Google Scholar. A few duplicates were manually removed due to differences in citation standards between sources. The articles were screened independently by BKH and HD using the predetermined inclusion criteria. A third researcher, YD and ATG independently resolved disagreements between the two writers. The data was extracted and evaluated independently by BKH, GGD and HD. First author, year of publication, year of study, location (region), co-infection, co-infection size, and co-infection event were among the collected data from eligible papers.

Quality Assessment

A comprehensive search for all possibly relevant articles and the application of precise, repeatable criteria for article selection were two of the tactics we used to reduce bias and random error. An established systematic methodology that complies with evidence-based methodological standards was followed in evaluating research designs and study characteristics, the synthesis of data, and the interpretation of the findings. To choose which papers to include and omit from the review, BK and HD examined the titles, abstracts, and full-text publications. After that, the articles are evaluated to see if they meet the specified eligibility requirements. BK assessed quality using the appraisal tool for cross-sectional studies (AXIS tool) (Supplementary File 2). There are twenty items on this checklist [32, 33].

Data Synthesis and Statistical Analysis

For analysis, the extracted data was loaded into R software version 4.4.1 from an Excel spreadsheet. Heterogeneity and heterogeneity quantification were determined by using Cochran’s Q-test and I² index, respectively. We considered the I² values of 25, 50, and 75% as low, medium and high heterogeneity, respectively [34–36]. The tau statistics (τ²) was used to assess the variance of the effect size estimates across the study population. Based on the heterogeneity assessment result, we used Der Simonian and Laird’s random-effects method (if the p-value of the Q test was < 0.05 and I² was > 50%). The random-effects model with the DerSimonian-Laird technique of selection [36, 37] was used to calculate the pooled prevalence and 95% confidence intervals (95% CIs). In contrast to a fixed-effect model, the random procedure includes an additional variance component to account for variability between studies (heterogeneity) as well as within-study variance due to sampling error [38]. Using the DerSimonian and Laird model, subgroup analyses were conducted for the prevalence of MDR-TB in the context of co-infections, taking into account publication year, geographic areas (northern, southern, central, and eastern Ethiopia), and various co-infections [39]. Next, funnel plot diagrams were used to visualise the presence of publication bias and the effects of small studies [40]. We used a meta-regression analysis to find possible sources. A p-value less than 0.05 (P < 0.05) was considered significant in all analyses [41].

Results

Search Result

Initially, a total of 6461 articles were identified from the included databases, of which 563 were duplicates. 5477 were filtered based on the publication region because the publications were inappropriate or systematic reviews and meta-analyses. The remaining 379 were deemed irrelevant to our review and were removed based on their titles and abstracts. After utilizing predetermined criteria to evaluate eligibility, the remaining 44 full-text articles were examined; 28 articles were eliminated for the following reasons: 9 did not meet the review’s specified emphasis, 9 lacked complete information, and 11 excluded important details about prevalence. Finally, 15 articles were included for this meta-analysis and systematic review (Fig. 1).

Fig. 1.

PRISMA flowchart for the selection of studies on the MDR-TB in the context of co-infection in Ethiopia

Study Characteristics

Table 1 displays the characteristics of the 15 articles included in this systematic review and meta-analysis. Every article included in this review was conducted in Ethiopia’s Central, Northern, Eastern, Western, and Southern regions. The researches were released in publications from 2015 to 2024. Twenty-two prevalence reports of MDR-TB in the context of co-infections were extracted from the 15 reports and included in the analysis. Of these publications, results for MDR-TB in the context of HIV co-infection were reported in all 15 articles, while results for MDR-TB in the context of diabetes co-infection were reported in only seven articles as indicated in Table 1.

Table 1.

Descriptive summary of the included studies on the prevalence of MDR-TB in the context of co-infections in Ethiopia

ID	Author	Study location	Co-infection	Total	Event	Event rate
1	Seid et al. [27]	Northern Ethiopia	HIV	30	5	0.167
2	Amin et al. [25]	Eastern Ethiopia	HIV	58	9	0.155
3	Amin et al. [25]	Eastern Ethiopia	Diabetic	30	5	0.167
4	Mekonnen et al. [28]	Northern Ethiopia	HIV	28	2	0.071
5	Mulisa et al. [26]	Central Ethiopia	HIV	72	17	0.236
6	Brhane et al. [42]	Eastern Ethiopia	HIV	8	3	0.375
7	Gobena et al. [43]	Southern Ethiopia	HIV	73	5	0.068
8	Gobena et al. [43]	Southern Ethiopia	Diabetic	59	8	0.136
9	Alene et al. [44]	Northern Ethiopia	HIV	242	51	0.211
10	Alene et al. [44]	Northern Ethiopia	Diabetic	242	5	0.021
11	Welekidan et al. [45]	Northern Ethiopia	HIV	41	8	0.195
12	Welekidan et al. [45]	Northern Ethiopia	Diabetic	8	2	0.25
13	Mesfin et al. [24]	Northern Ethiopia	HIV	199	71	0.357
14	Seid et al. [23]	Northern Ethiopia	HIV	36	5	0.139
15	Wakjira et al. [22]	Central Ethiopia	HIV	131	34	0.26
16	Wakjira et al. [22]	Central Ethiopia	Diabetic	41	5	0.122
17	Badgeba et al. [11]	Southern Ethiopia	HIV	5	2	0.4
18	Badgeba et al. [11]	Southern Ethiopia	Diabetic	5	1	0.2
19	Bade & Mega, [46]	Southern Ethiopia	HIV	200	44	0.22
20	Bade & Mega, [46]	Southern Ethiopia	Diabetic	200	9	0.045
21	Ambaye & Tsegaye, [47]	Northern Ethiopia	HIV	29	22	0.759
22	Kassa et al. [48]	Central Ethiopia	HIV	451	118	0.262

Meta-Analysis

A total of 22 reports were considered in the current meta-analysis. The pooled prevalence of MDR-TB in the context of co-infection in Ethiopia was 20% (95% CI: 14, 26). The reported pooled prevalence of MDR-TB in the context of co-infections showed significant heterogeneity amongst the studies (tau² = 0.0185, H² =19.73, I² = 94.93%, Q-test = 349.6061, df = 21, p ≤ 0.001) (Fig. 2).

Fig. 2.

Forest plot showing the pooled prevalence of MDR-TB in the context of co-infections in Ethiopia

Quality Assessment Result

In this review, a range of studies, with quality ratings from low to moderate, was evaluated. Notably, all studies employed a clear sample size estimation method. In the current meta-analysis, all articles used the random sampling method as outlined by [49]. Additionally, all 15 studies (100%) obtained a sample frame from a population closely resembling the target or reference population. Of these, 13 studies (86.7%) met six of the 20 key criteria, including aims/objectives, definition of the target/reference population, internal consistency of results, justification of findings, sample size justification, and appropriate methodological techniques. Conflicts of interest and descriptions of statistical methods were also addressed.

Subgroup Meta-Analysis

The results of a subgroup analysis based on geographical location are shown in Table 2. Accordingly, the highest pooled prevalence of MDR-TB in the context of co-infection was found in Northern Ethiopia (22.9% (95% CI: 13.3; 32.6)), whereas the lowest was found in Southern Ethiopia (14% (95% CI: 15.7; 26.6)) (Table 2). Furthermore, a high degree of heterogeneity between the various groups was shown by the subgroup analysis’s heterogeneity results (tau² = 0.0180, I² = 94%, Q-test = 349.61, df = 21, p ≤ 0.001) (Supplementary Fig. 1).

Table 2.

Subgroup analysis for comparing the prevalence of MDR-TB in different study locations

Study location	K	Prevalence (95% CI)	tau2	tau	p-value
Northern Ethiopia	9	22.9 [13.3; 32.6]	96%	0.0180	0.01
Eastern Ethiopia	3	20.0 [1.71; 38.3]	0%	0.0180	0.46
Central Ethiopia	4	22.1 [84.0; 35.9]	55%	0.0180	0.09
Southern Ethiopia	6	14.0 [15.7; 26.6]	85%	0.0180	0.01
Overall	22	20	94%	0.0180	0.01

K = Number of studies, CI = Confidence interval

The result indicated in Table 3 showed a subgroup analysis based on publication years. Studies conducted between 2021 and 2024 had a greater pooled prevalence of MDR-TB in the context of co-infections (24.6%; 95% CI: 15.1; 34.0) than Studies conducted between 2015 and 2020 (17.4%; 95% CI: 10.2; 24.5) (Table 3) (Supplementary Fig. 2).

Table 3.

Subgroup analysis for comparing the prevalence of MDR-TB by publication years

Publication year	K	Prevalence (95% CI)	tau2	tau	p-value
Studies conducted between 2015–2020 (A)	13	17.4 [10.2; 24.5]	95%	0.0144	0.01
Studies conducted between 2021–2024 (B)	9	24.6 [15.1; 34.0]	86%	0.0144	0.01
Overall	22	20	94%	0.0180	0.01

K = Number of studies, CI = Confidence interval

The subgroup analysis, which was carried out based on different co-infections, was displayed in Table 4. Among the co-infections, the highest pooled prevalence of MDR-TB was recorded from HIV-infected individuals (23.2%; 95% CI: 18.3, 28.0) as compared to diabetic patients (Table 4) (Supplementary Fig. 3).

Table 4.

Subgroup analysis for comparing the prevalence of MDR-TB between different co-infections

Co-infections	K	Prevalence (95% CI)	tau2	tau	p-value
HIV	15	23.2 [18.3; 28.0]	87%	0.0062	0.01
Diabetes	7	10 [3; 17.3]	66%	0.0062	0.01
Overall	22	19	94%	0.0062	0.01

K = Number of studies, CI = Confidence interval

Publication Bias

Publication bias and small study effects were assessed using funnel plot observation and Egger’s regression test, respectively. The result comparing the log odds of effects against their standard errors, indicates no evidence of slope asymmetry, with a p-value of 0.5242 (Fig. 3). In addition. the egger regression test result is non-significant (p = 0.8989), so there is no significant publication bias between the included studies (Table 5).

Fig. 3.

Funnel plot showing small study effects

Table 5.

Publication bias assessment

Standard effect	Coefficient	z-value	p-value	95% CI
Slope	-1.29	-0.64	0.5242	-1.96, -0.64
Bias	-0.08	-0. 13	0.8989	-0.12, -0.03

Meta-Regression

In this systematic review and meta-analysis, meta-regression was conducted to identify the potential factors by considering different co-infections, publication years and study locations. There was a statistically significant association (p < 0.05) observed between the occurrence of MDR-TB with co-infections (Table 6).

Table 6.

Final multivariable Meta-regression model

Variables	Coefficient	P-value	95% CI
Region
Central Ethiopia	Ref.
Eastern Ethiopia	-0.064	0.645	-0.33, 0.20
Northern Ethiopia	0.052	0.545	-0.11, 0.22
Southern Ethiopia	-0.006	0.952	-0.20, 0.19
Pub year
A	Ref.
B	0.118	0.147	-0.04, 0.28
Co-Infection
Diabetes	Ref.
HIV	0.157	0.047	-0.15, 0.25

Discussion

The prevalence of MDR-TB is rising worldwide, with the majority of cases attributed to a combination of poor patient adherence and prescription errors [50]. Despite the high TB/HIV and MDR-TB prevalence in Ethiopia, there remains a significant information gap regarding drug-resistant TB in the country [51]. The objective of this study was to provide a comprehensive view of the current published evidence and estimate the burden of MDR-TB in the context of co-infections in Ethiopia.

In this systematic review and meta-analysis, which included 16 original studies, the pooled prevalence of MDR-TB in the context of co-infections in Ethiopia was found to be 20% (95% CI: 14, 26). The current finding is higher than previous systematic review and meta-analysis report conducted in Ethiopia by Asgedom et al. [52], Reta et al. [53], Eshetie et al. [51], Diriba et al. [54], Mesfin et al. [55], Eshetie et al. [56], and Alene et al. [57] who reported pooled prevalence of MDR-TB 1.4%, 2.64%, 2%, 9%, 1.24%, 1.75% and 7.5%, respectively. Similarly, the current finding is higher than the reports of Molla et al. [58] in East Africa, who reported a pooled prevalence of 1.24%. However, the current finding was lower when compared with the pooled prevalence report of 22% by Teweldemedhin et al. [59] in a systematic review and meta-analysis study in Ethiopia. The variation in the pooled prevalence rates of MDR-TB between the different studies might be due to differences in the geographical location, number of studies included, study population, drug resistance profiles, and diagnostic techniques [60].

The prevalence of MDR-TB in the context of HIV co-infection was found to be 23.2% (95% CI: 18.3, 28.0). This result is higher than the previous reports in Ethiopia by Girum et al. [13], Mesfin et al. [55], Dessalegn et al. [61], Mersha et al. [62], Bade and Mega [46], and Mulu et al. [63] who reported pooled prevalence of MDR TB in the context of HIV co-infection 1.17%, 2.28%, 18.4%, 15.3%, 22%, 19.6%, respectively. Furthermore, the current result is lower than the prevalence reported by Wakjira et al. [22] (83%) and Mesfin et al. [24] (56.6%), and almost equal to the prevalence reported by Seid et al. [23] (19.8%). Furthermore, in the current study, the pooled prevalence of MDR-TB in the context of diabetes co-infection was 10% (95% CI: 3, 17.3). This result is higher than the pooled prevalence reported by Workneh et al. [64] and Bade and Mega [46], who reported a pooled prevalence of 8.3% and 4.5%, respectively. Additionally, this result is lower than the prevalence reported by Tegegne et al. [65] (97%) and Wakjira et al. [22] (12%) and equal to the prevalence reported by Adane et al. [66] (10%). The differences in prevalence results of MDR-TB in the context of co-infections, such as HIV and diabetes, can be attributed to various factors: population characteristics (including demographic and health status), sample size and study design, co-infection burden, and referral bias. Additionally, HIV infection increases the risk of developing MDR-TB by compromising the immune system, which impairs the body’s ability to combat tuberculosis pathogens. Furthermore, individuals living with HIV are more susceptible to MDR-TB infections and may have a higher likelihood of developing primary resistance to tuberculosis drugs [67–69]. Diabetes significantly contributed to the risk of treatment failure in tuberculosis patients and the emergence of MDR-TB in patients with tuberculosis and diabetes comorbidity [70, 71].

Subgroup analysis based on geographical location reported that the highest prevalence of MDR-TB in the context of co-infections was found in Northern Ethiopia (22.9%). This result is higher than the prevalence reported by Bez et al. [72], Mohammed et al. [73], and Mehari et al. [74] who reported a prevalence of 3.4%, 12.2% and 19.4%, respectively in studies conducted in Northern Ethiopia. The lowest pooled prevalence was found in Southern Ethiopia at 14%. This result is lower than the prevalence reported by Mehari et al. [74] (22.6%) and higher than that reported by Mesfin et al. [24] (0%). The pooled prevalence in Eastern Ethiopia was found to be 20%, which is consistent with the prevalence reported by Mehari et al. [74] (20%); however, the current finding is higher than the prevalence reports of Mesfin et al. [24] (2.2%). Moreover, the pooled prevalence in Central Ethiopia was found to be 22.1%, which is lower than the prevalence reported by Mesfin et al. [24] (93.3%) and higher than that reported by Mehari et al. [74] (13.9%). These variations in the prevalence across different locations may be attributed to differences in the number of studies included in the meta-analysis. In addition, the substantial difference between the regions might be due to the difference in the socio-cultural and demographic characteristics of the population [75].

Limitations of the Study

Although the current study is the first to summarize different co-infections prevalence of MDR-TB in Ethiopia over a 10-year period, we acknowledge its limitations. The number of prevalence studies on MDR-TB in most areas of the country was limited, with only a few meeting our eligibility criteria. Prevalence data on MDR-TB in the context of co-infections from Western Ethiopia was not included in our study due to the absence of eligible published work from these regions. Thus, the reported pooled prevalence may differ if data from these regions were available.

Conclusion

The current systematic review and meta-analysis of MDR-TB and co-infections in Ethiopia reveals important obstacles to clinical management and public health. The pooled prevalence of MDR-TB in the context of co-infections in Ethiopia was found to be 20%. In addition, MDR-TB in the context of HIV co-infection has the highest prevalence, around 23.2%, then diabetes co-infection. Therefore, we recommend future research focusing on understudied areas of Ethiopia to offer a more comprehensive grasp of MDR-TB and associated co-infections throughout the country. Additionally, there is a need for more studies that explore a broader spectrum of co-infections beyond HIV and diabetes.

Electronic Supplementary Material

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Abbreviations

DR-TB

Drug resistance Tuberculosis

EMB

Ethambutol

HIV

Human immunodeficiency virus

INH

Isoniazid

MDR-TB

Multi drug resistance

PZA

Pyrazinamide

RIF

Rifampin

TB

Tuberculosis

WHO

World health organization

Author Contributions

The contribution of the authors to the manuscript as follows, Conceptualization and design of the study: BKH, GGD, and HD; Acquisition of data, or analysis and interpretation of data: BKH, YD, ATG, GGD and HD; Software, Supervision, Investigation, Visualization and Validation: YD, ATG, GGD and HD; Drafting the article or revising it critically for important intellectual content: BKH, YD, ATG, GGD and HD. All authors read and approved the final version of the manuscript to be submitted.

Funding

No funding was received for this research.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethical Approval

Ethical approval is not applicable. No animal or human experimentation was undertaken.

Consent for Publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.